Wound healing impairment in diabetic patients represents a particular challenging clinical problem to which no efficacious treatment regimens exist. Endothelial cell dysfunction is a significant contributor to impaired wound healing. A cardinal feature of endothelial dysfunction in diabetes is hyperglycemia- mediated superoxide anion overproduction by the mitochondrial electron transport chain with resultant oxidative stress. Our preliminary data suggest that increased superoxide in diabetes appears to impair the functions of three endothelial factors on wound healing - tetrahydrobiopterin (BH4) and endothelial nitric oxide synthase (eNOS), endothelial morphogen sonic hedgehog (SHH), and endothelial progenitor cells (EPCs). Yet remarkably little is known about how superoxide-induced oxidative stress regulates these factors central to wound repair. Such knowledge is critical for successfully modulating endothelial function to improve wound healing in diabetes. For example, discovery of the defects in diabetic EPC and the means to correct them could lead to autologous cell therapies for diabetic wounds. This project will focus on oxidative stress-induced endothelial dysfunction in diabetic wound repair, thus filling a significant gap in such knowledge. We hypothesize that oxidative stress in diabetic wounds is a common cause for the dysfunction of the three repair mediators - eNOS, sonic hedgehog and endothelial progenitor cells, resulting in impaired healing. To test this hypothesis, we will pursue three specific aims using streptozotocin-induced type 1 diabetic mice. In aim 1, we will determine how oxidative stress impairs cutaneous eNOS function, focusing on the role of its essential cofactor BH4. In Aim 2, we will determine the mechanism through which sonic hedgehog regulates diabetic wound healing under oxidative stress. In Aim 3, we will determine how endothelial progenitor cells become dysfunctional in type 1 diabetes. The major significance of the proposed studies is that it will for the first time determine how superoxide regulates endothelial function and wound repair in an integrated fashion, which may provide new knowledge regarding an interconnected mechanism that affects wound healing in type 1 diabetes, thus addressing a fundamental issue in system biology and pathophysiology of wound healing.